The interference of expression of specific genes following the introduction of double-stranded RNAs (dsRNAs) of corresponding sequence has been observed in a variety of organisms. Initially described as the post-transcriptional gene silencing (PTSG) of transgenes in transgenic plants, similar dsRNA-dependent gene silencing has now been observed in protozoa, fungi, nematodes, insects, and mammals, and the phenomenon is now generally referred to as “RNA interference.”
RNA interference (RNAi), which has been defined as the “the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene” (Elbashir, et al., Nature 411: 494-498 (2001)), has also been described as “the process whereby dsRNA induces the sequence-specific degradation of homologous mRNA” (Chiu & Rana. Molecular Cell 10:549-561 (2002)). Many of the mechanistic details of RNAi first came to light from studies in which long dsRNAs matching the sequences of specific target gene transcripts were introduced into the nematode worm, Caenorhabditis elegans (Fire et al., Nature 391:806-811 (1998)). These early studies inspired many more experiments to be conducted in a variety of organisms, and it is now clear that homologous machinery for RNAi is widely distributed among eukaryotic organisms, including mammals. Although initial attempts to provoke RNAi in mammals and mammalian cells with long dsRNAs failed, it was later determined the failures occurred because such RNA molecules activate an antiviral response that leads to a general inhibition of translation and ultimately cell death. Subsequent studies in a variety of non-mammalian species demonstrated that long dsRNAs introduced into cells are enzymatically cleaved into shorter duplexes comprising two complementary single-stranded RNAs of 21-25 nucleotides (see Sharp, Genes & Dev. 15:485-490 (2001), and references therein). More recent studies have demonstrated that, while dsRNAs of 30 basepairs or more elicit the aforementioned antiviral response, RNA duplexes comprising two complementary single-strands of 21 nucleotides each, which pair to form a 19 basepair duplexed region with two nucleotide 3′ overhangs (so called small or short interfering RNAs (siRNAs)), can mediate RNAi in cultured mammalian cells without evoking an antiviral response (Elbashir et al., Nature 411:494-498 (2001)). Additionally, when appropriately targeted via their nucleotide sequence, these siRNAs can specifically suppress the expression of both endogenous genes and heterologous transgenes, of corresponding sequence. Even more recent studies have demonstrated that while double-stranded siRNAs are very effective at mediating RNAi in a variety of cell types, short, single-stranded, hairpin-shaped RNAs can also mediate RNAi, presumably because they are processed into siRNAs by cellular enzymes (Sui et al., Proc. Natl. Acad. Sci. U.S.A. 99:5515-5520 (2002); Yu et al., Proc. Natl. Acad. Sci. U.S.A. 99:6047-6052 (2002); and Paul et al., Nature Biotech. 20:505-508 (2002)). This discovery has significant and far-reaching implications since the production of such small hairpin RNAs (shRNAs) can be readily achieved in vivo by transfecting cells with transcription vectors bearing short inverted repeats separated by a small number of (e.g., six) nucleotides. Additionally, if features are included to ensure the stability of the transcription plasmid, or direct the integration of the transcription cassette into the host cell genome, the RNAi induced by the encoded shRNAs, can be made stable and heritable.
Since it was first exploited to silence specific genes in C. elegans, RNAi has become an irreplaceable tool for molecular, cellular and developmental biologists seeking to discover the functions of specific genes. Although few of the molecular mechanisms of RNAi are known in detail, it is clear that the degradative process involves the assembly of a multisubunit ribonucleoprotein nuclease complex, known as RISC (for RNA-induced silencing complex), which is somehow guided by the antisense strand of an siRNA to a complementary target sequence in a mature RNA transcript, where it catalyzes the cleavage of the targeted transcript. It is also clear that all that is necessary to target a transcript for degradation is that the sequence of the antisense strand of an siRNA be complementary to that of a “target sequence” in the transcript to be degraded. Empirical studies have shown, however, that not all target sequences are equivalent. That is, siRNAs corresponding to different target sequences within the same transcript can exhibit significantly different efficiencies in directing the degradation of the same transcript. Furthermore, at present it is impossible to predict which target sequences will prove to be most effective targets. Practically, if one wants to efficiently silence a particular gene by RNA interference, one must empirically determine which sequences make the best targets by designing and testing siRNAs corresponding to different target sequences. This process is both cumbersome and time consuming.
In all taxa exhibiting RNAi, siRNAs corresponding to a specific target sequence in a gene or transgene (primary siRNAs or trigger siRNAs), evoke a “primary” RNAi response, wherein the targeted transcript is cleaved in the region of nucleotide sequence complementary to the antisense strand of the siRNA. However, in C. elegans and plants, a “secondary” RNAi response is observed, wherein “secondary” siRNAs are produced that direct cleavage of the target transcript in regions outside of the original target sequence. In C. elegans, these secondary cleavages occur at sites exclusively 5′ of the primary siRNA target sequence, but in plants, these secondary cleavages occur at sites either 5′ or 3′ of the primary siRNA target sequence. Additionally, if the regions of nucleotide sequence in which secondary RNAi cleavages occur are homologous to other transcripts within the cell, the secondary RNAi response can lead to silencing of transcripts containing highly similar nucleotide sequences, that were not initially targeted by the trigger siRNA. The observed secondary RNAi response has been termed “transitive RNAi,” because the sites of cleavage during the secondary response transit along the originally targeted transcript away from the primary target to adjacent regions, or the silencing transits to transcripts that were not initially targeted during the primary RNAi response.
Although the specific mechanisms operating behind the phenomenon of transitive RNAi remain to be elucidated, the taxa that exhibit transitive RNAi also appear to be able to “amplify” the gene silencing response induced by primary siRNAs. Additionally, it has been shown that in Arabadopsis and C. elegans, transitive RNAi requires the action of putative RNA-dependent RNA polymerases (RdRPs) (Dalmay et al., Cell 101:543-553 (2000) and Sijen et al., Cell 107:465-476 (2001)). Consequently, it has been hypothesized that in C. elegans, transitive RNAi involves an amplification step catalyzed by RdRP, whereby the antisense strand of siRNAs serve as primers for synthesis of dsRNAs by RdRP, using the targeted transcript as the template, and the nascent dsRNAs are subsequently cleaved by an endonuclease (Dicer) to produce secondary siRNAs (Sijen et al., Cell 107:465-476 (2001). If the nascent dsRNA so synthesized contains nucleotide sequences highly similar to nucleotides sequences in RNA transcripts not targeted by the initial siRNA during a primary RNAi response, the dsRNA corresponding to these nucleotide sequences will be cleaved to form the “secondary” siRNAs necessary to target these alternate transcripts and transit the silencing to the gene products encoded by these alternate transcripts. This hypothesis is consistent with the observation that, in some studies, siRNAs designed to target a particular member of a gene family, ultimately induced silencing of the entire family of genes.
Although RNAi has proven to be a remarkably powerful tool for investigating gene function in a variety of taxa, as mentioned above, Tuschl and colleagues only recently discovered that 21-nucleotide siRNAs could be used for studying gene function in mammalian cells without evoking a general antiviral response (Elbashir et al., Nature 411:494-498 (2001)). Unfortunately, once a gene is selected for siRNA-induced silencing, the choice of which sequences to target by siRNAs is somewhat unclear. Towards this end, Holen and colleagues investigated the efficacy of siRNAs targeted to different positions in the transcript of human coagulation trigger Tissue Factor (hTF) in a variety of human cell types in culture (See Holen et al., Nucleic Acids Res. 30:1757-1766 (2002)). In this study several siRNAs corresponding to several target sequences located in hTF transcripts were synthesized and tested for their ability to induce silencing of the hTF gene. Of the several siRNAs synthesized and tested only a few resulted in a significant reduction in expression of hTF, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Further, siRNAs targeting different sites in the hTF mRNA demonstrated striking differences in their ability to silence the expression of hTF. Although, strong positional effects were seen with the siRNAs tested, and regions of high GC content seem to be targeted less efficiently than those of low GC content, Holen and coworkers concluded that the factors determining the differences in siRNA efficiency remain unclear, and that susceptible RNAi target sites in some human genes may be rare.
From a practical perspective, the results of Holen and colleagues suggest that it is difficult, if not impossible to predict, a priori, what sequences to target in a gene to target with siRNAs to induce efficient silencing by RNAi. In addition, there is a growing body of evidence that specific siRNAs selected to silence particular genes may produce unwanted and unanticipated “off-target” effects—altering the expression of untargeted RNA transcripts. Jackson and colleagues recently published the results of a study of off-target gene regulation conducted using a gene expression profiling technique to characterize the specificity of gene silencing by siRNAs in cultured human cells. Their results provide clear evidence that treatment of cells with siRNAs corresponding to different sequences within the same RNA transcript may result in different, but reproducible, off-target silencing effects, at least some of which may be due to partial sequence homology between the affected transcripts and either the sense or the antisense strand of the siRNA employed. They conclude that it may be difficult to select an siRNA sequence that will be absolutely specific for the target of interest (Jackson et al., Nature Biotech. 21:635-637 (2003)).
Recent advances in genomics, especially with the completion of the human genome sequence, have lead to the discovery of numerous novel genes of unknown function. Unfortunately, advances in our ability to sequence genomes, and identify novel genes within them have far outstripped our ability to determine the function of the gene products of these novel genes. Classically, gene function has been addressed in vivo by two distinct approaches: overexpression of the gene product and underexpression of the gene product.
High-throughput methodology in biotechnology is largely responsible for the recent explosive growth of knowledge in genomics and proteomics—two specialty fields that are relatively new to the larger field of molecular biology. In the realm of pharmaceutical research and development, high-throughput technologies, genomics and proteomics, have had a profound impact on therapeutic drug development (Kennedy. EXS. 89:1-10 (2000)). Such technologies have issued in a “new millennium” of drug discovery (Cunningham. J. Pharmacol. Toxicol. Methods. 44:291-300 (2000)), and have provided a catalyst for change in drug discovery paradigms (Hanke. J. Law Med. Ethics. 28(4 Suppl): 15-22 (2000)). Such technologies have the potential for greatly increasing the speed of drug development, and for reducing the associated costs—both of these factors being critically important given the current economic and social climate. Clearly, significant improvements in the ability of research scientists to (a) selectively overexpress specific target genes, (b) selectively block the expression of specific target genes, (c) screen large numbers of target genes for their cellular functions, and (d) ultimately determine how overexpression or underexpression of specific target genes affect desired outcomes in mammalian cells, will be of benefit to society.
Traditionally, RNAi has been applied to investigate the function of genes in a one-target-at-a-time mode. This approach has proven very useful in analyzing the function of a limited number of genes in the model organisms C. elegans and D. melanogaster. The use of microarray-based RNAi technology using siRNAs should greatly facilitate the investigation of functions for hundreds or thousands of mammalian genes simultaneously in a parallel fashion. However, given the findings of Holen et al. and Jackson et al., discussed above, the choice of what specific sequence within an mRNA to target with siRNAs of corresponding sequence is completely unclear. Further, although microarray-based RNAi technology will perhaps allow the empirical identification of sequences that will serve as ideal targets of RNAi, the process of synthesizing and testing numerous siRNAs is laborious and costly.
While RNAi holds much promise for high-throughput analyses designed to determine gene function through the silencing of large numbers of genes, the method is not without complications and challenges. Perhaps the most fundamental challenge is how to pick a target sequence within the target transcript that will allow for the efficient silencing of the target gene, along with minimal unintended and undesired off-target effects. Despite numerous studies in which siRNAs have been employed to induce gene silencing, no definitive rules have evolved to assist researchers in picking the most effective sequences to target within a given transcript. Although there are general guidelines to help researchers narrow their choices for target sequences, researchers must still use a trial and error approach to empirically determine what individual siRNAs work best, and what siRNAs have minimal off-target effects. Given these limitations and the many potential and varied applications of RNAi, there is a clear need for alternative approaches and techniques for altering gene expression by siRNAs, especially with regards to high-throughput applications.